Thyroid hormone analogs and methods of use

ABSTRACT

Disclosed are methods of treating subjects having conditions related to angiogenesis including administering an effective amount of a polymeric form of thyroid hormone, or an antagonist thereof, to promote or inhibit angiogenesis in the subject. Compositions of the polymeric forms of thyroid hormone, or thyroid hormone analogs, are also disclosed.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/943,072, filed Sep.15, 2004, which claims priority to U.S. Ser. No. 60/502,721, filed Sep.15, 2003, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to thyroid hormone, thyroid hormone analogs andderivatives, and polymeric forms thereof. Methods of using suchcompounds, and pharmaceutical compositions containing same are alsodisclosed. The invention also relates to methods of preparing suchcompounds.

BACKGROUND OF THE INVENTION

Thyroid hormones, L-thyroxin (T4) and L-triiodothyronine (T3), regulatemany different physiological processes in different tissues invertebrates. Most of the actions of thyroid hormones are mediated by thethyroid hormone receptor (“TR”), which is a member of the nuclearreceptor superfamily of ligand-activated transcription regulators. Thissuperfamily also includes receptors for steroid hormones, retinoids, and1,25-dihydroxyvitamin D3. These receptors are transcription factors thatcan regulate expression of specific genes in various tissues and aretargets for widely used drugs, such as tamoxifen, an estrogen receptorpartial antagonist. There are two different genes that encode twodifferent TRs, TRα and TRβ. These two TRs are often co-expressed atdifferent levels in different tissues. Most thyroid hormones do notdiscriminate between the two TRs and bind both with similar affinities.

Gene knockout studies in mice indicate that TRβ plays a role in thedevelopment of the auditory system and in the negative feedback ofthyroid stimulating hormone by T3 in the pituitary, whereas TRαmodulates the effect of thyroid hormone on calorigenesis and on thecardiovascular system. The identification of TR antagonists could playan important role in the future treatment of hypothyroidism. Suchmolecules would act rapidly by directly antagonizing the effect ofthyroid hormone at the receptor level, a significant improvement forindividuals with hypothyroidism who require surgery, have cardiacdisease, or are at risk for life-threatening thyrotoxic storm.

Thus, there remains a need for the development of compounds thatselectively modulate thyroid hormone action by functioning asisoform-selective agonists or antagonists of the thyroid hormonereceptors (TRs) would prove useful for medical therapy. Recent effortshave focused on the design and synthesis of thyroid hormone (T3/T4)antagonists as potential therapeutic agents and chemical probes. Thereis also a need for the development of thyromimetic compounds that aremore accessible than the natural hormone and have potentially usefulreceptor binding and activation properties.

It is estimated that five million people are afflicted with chronicstable angina in the United States. Each year 200,000 people under theage of 65 die with what is termed “premature ischemic heart disease.”Despite medical therapy, many go on to suffer myocardial infarction anddebilitating symptoms prompting the need for revascularization witheither percutaneous transluminal coronary angioplasty or coronary arterybypass surgery. It has been postulated that one way of relievingmyocardial ischemia would be to enhance coronary collateral circulation.

Correlations have now been made between the anatomic appearance ofcoronary collateral vessels (“collaterals”) visualized at the time ofintracoronary thrombolitic therapy during the acute phase of myocardialinfarction and the creatine kinase time-activity curve, infarct size,and aneurysm formation. These studies demonstrate a protective role ofcollaterals in hearts with coronary obstructive disease, showing smallerinfarcts, less aneurysm formation, and improved ventricular functioncompared with patients in whom collaterals were not visualized. When thecardiac myocyte is rendered ischemic, collaterals develop actively bygrowth with DNA replication and mitosis of endothelial and smooth musclecells. Once ischemia develops, these factors are activated and becomeavailable for receptor occupation, which may initiate angiogenesis afterexposure to exogenous heparin. Unfortunately, the “natural” process bywhich angiogenesis occurs is inadequate to reverse the ischemia inalmost all patients with coronary artery disease.

During ischemia, adenosine is released through the breakdown of ATP.Adenosine participates in many cardio-protective biological events.Adenosine has a role in hemodynamic changes such as bradycardia andvasodilation, and adenosine has been suggested to have a role in suchunrelated phenomena as preconditioning and possibly the reduction inreperfusion injury (Ely and Beme, Circulation, 85: 893 (1992).

Angiogenesis is the development of new blood vessels from preexistingblood vessels (Mousa, S. A., In Angiogenesis Inhibitors and Stimulators:Potential Therapeutic Implications, Landes Bioscience, Georgetown, Tex.;Chapter 1, (2000)). Physiologically, angiogenesis ensures properdevelopment of mature organisms, prepares the womb for egg implantation,and plays a key role in wound healing. The development of vascularnetworks during embryogenesis or normal and pathological angiogenesisdepends on growth factors and cellular interactions with theextracellular matrix (Breier et al., Trends in Cell Biology 6:454-456(1996); Folkman, Nature Medicine 1:27-31 (1995); Risau, Nature386:671-674 (1997). Blood vessels arise during embryogenesis by twoprocesses: vasculogenesis and angiogenesis (Blood et al., Bioch.Biophys. Acta 1032:89-118 (1990). Angiogenesis is a multi-step processcontrolled by the balance of pro- and anti-angiogenic factors. Thelatter stages of this process involve proliferation and the organizationof endothelial cells (EC) into tube-like structures. Growth factors suchas FGF2 and VEGF are thought to be key players in promoting endothelialcell growth and differentiation.

Control of angiogenesis is a complex process involving local release ofvascular growth factors (P Carmeliet, Ann NY Acad Sci 902:249-260,2000), extracellular matrix, adhesion molecules and metabolic factors (RJ Tomanek, G C Schatteman, Anat Rec 261:126-135, 2000). Mechanicalforces within blood vessels may also play a role (O Hudlicka, Molec CellBiochem 147:57-68, 1995). The principal classes of endogenous growthfactors implicated in new blood vessel growth are the fibroblast growthfactor (FGF) family and vascular endothelial growth factor (VEGF)(GPages, Ann NY Acad Sci 902:187-200, 2000). The mitogen-activated proteinkinase (MAPK; ERK1/2) signal transduction cascade is involved both inVEGF gene expression and in control of proliferation of vascularendothelial cells.

Intrinsic adenosine may facilitate the coronary flow response toincreased myocardial oxygen demands and so modulate the coronary flowreserve (Ethier et al., Am. J. Physiol., H131 (1993) demonstrated thatthe addition of physiological concentrations of adenosine to humanumbilical vein endothelial cell cultures stimulates proliferation,possibly via a surface receptor. Adenosine may be a factor for humanendothelial cell growth and possibly angiogenesis. Angiogenesis appearsto be protective for patients with obstructive blood flow such ascoronary artery disease (“CAD”), but the rate at which blood vesselsgrow naturally is inadequate to reverse the disease. Thus, strategies toenhance and accelerate the body's natural angiogenesis potential shouldbe beneficial in patients with CAD.

Similarly, wound healing is a major problem in many developing countriesand diabetics have impaired wound healing and chronic inflammatorydisorders, with increased use of various cyclooxygenase-2 (CoX2)inhibitors. Angiogenesis is necessary for wound repair since the newvessels provide nutrients to support the active cells, promotegranulation tissue formation and facilitate the clearance of debris.Approximately 60% of the granulation tissue mass is composed of bloodvessels which also supply the necessary oxygen to stimulate repair andvessel growth. It is well documented that angiogenic factors are presentin wound fluid and promote repair while antiangiogenic factors inhibitrepair. Wound angiogenesis is a complex multi-step process. Despite adetailed knowledge about many angiogenic factors, little progress hasbeen made in defining the source of these factors, the regulatory eventsinvolved in wound angiogenesis and in the clinical use of angiogenicstimulants to promote repair. Further complicating the understanding ofwound angiogenesis and repair is the fact that the mechanisms andmediators involved in repair likely vary depending on the depth of thewound, type of wound (burn, trauma, etc.), and the location (muscle,skin, bone, etc.). The condition and age of the patient (diabetic,paraplegic, on steroid therapy, elderly vs infant, etc) can alsodetermine the rate of repair and response to angiogenic factors. The sexof the patient and hormonal status (premenopausal, post menopausal,etc.) may also influence the repair mechanisms and responses. Impairedwound healing particularly affects the elderly and many of the 14million diabetics in the United States. Because reduced angiogenesis isoften a causative agent for wound healing problems in these patientpopulations, it is important to define the angiogenic factors importantin wound repair and to develop clinical uses to prevent and/or correctimpaired wound healing.

Thus, there remains a need for an effective therapy in the way ofangiogenic agents as either primary or adjunctive therapy for promotionof wound healing, coronary angiogenesis, or other angiogenic-relateddisorders, with minimum side effects. Such a therapy would beparticularly useful for patients who have vascular disorders such asmyocardial infarctions, stroke or peripheral artery diseases and couldbe used prophylactically in patients who have poor coronary circulation,which places them at high risk of ischemia and myocardial infarctions.

It is interesting to note that angiogenesis also occurs in othersituations, but which are undesirable, including solid tumour growth andmetastasis; rheumatoid arthritis; psoriasis; scleroderma; and threecommon causes of blindness—diabetic retinopathy, retrolental fibroplasiaand neovascular glaucoma (in fact, diseases of the eye are almost alwaysaccompanied by vascularization. The process of wound angiogenesisactually has many features in common with tumour angiogenesis. Thus,there are some conditions, such as diabetic retinopathy or theoccurrence of primary or metastatic tumors, where angiogenesis isundesirable. Thus, there remains a need for methods by which to inhibitthe effect of angiogenic agents.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that thyroid hormone,thyroid hormone analogs, and their polymeric forms, act at the cellmembrane level and have pro-angiogenic properties that are independentof the nuclear thyroid hormone effects. Accordingly, these thyroidhormone analogs and polymeric forms (i.e., angiogenic agents) can beused to treat a variety of disorders. Similarly, the invention is alsobased on the discovery that thyroid hormone analog antagonists inhibitthe pro-angiogenic effect of such analogs, and can also be used to treata variety of disorders.

Accordingly, in one aspect the invention features methods for treating acondition amenable to treatment by promoting angiogenesis byadministering to a subject in need thereof an amount of a polymeric formof thyroid hormone, or an analog thereof, effective for promotingangiogenesis. Examples of such conditions amenable to treatment bypromoting angiogenesis are provided herein and can include occlusivevascular disease, coronary disease, erectile dysfunction, myocardialinfarction, ischemia, stroke, peripheral artery vascular disorders, andwounds.

Examples of thyroid hormone analogs are also provided herein and caninclude triiodothyronine (T3), levothyroxine (T4),3,5-dimethyl-4-(4′-hydroy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or 3,5-diiodothyropropionic acid (DITPA), tetraiodothyroaceticacid (TETRAC), and triiodothyroacetic acid (TRIAC). Additional analogsare in FIG. 20 Tables A-D. These analogs can be conjugated to polyvinylalcohol, acrylic acid ethylene co-polymer, polylactic acid, or agarose.The conjugation is via covalent or non-covalent bonds depending on thepolymer used.

In one embodiment the thyroid hormone, thyroid hormone analogs, orpolymeric forms thereof are administered by parenteral, oral, rectal, ortopical means, or combinations thereof. Parenteral modes ofadministration include, for example, subcutaneous, intraperitoneal,intramuscular, or intravenous modes, such as by catheter. Topical modesof administration can include, for example, a band-aid.

In another embodiment, the thyroid hormone, thyroid hormone analogs, orpolymeric forms thereof can be encapsulated or incorporated in amicroparticle, liposome, or polymer. The polymer can include, forexample, polyglycolide, polylactide, or co-polymers thereof. Theliposome or microparticle has a size of about less than 200 nanometers,and can be administered via one or more parenteral routes, or anothermode of administration. In another embodiment the liposome ormicroparticle can be lodged in capillary beds surrounding ischemictissue, or applied to the inside of a blood vessel via a catheter.

Thyroid hormone, thyroid hormone analogs, or polymeric forms thereofaccording to the invention can also be co-administered with one or morebiologically active substances that can include, for example, growthfactors, vasodilators, anti-coagulants, anti-virals, anti-bacterials,anti-inflammatories, immuno-suppressants, analgesics, vascularizingagents, or cell adhesion molecules, or combinations thereof. In oneembodiment, the thyroid hormone analog or polymeric form is administeredas a bolus injection prior to or post-administering one or morebiologically active substance.

Growth factors can include, for example, transforming growth factoralpha (TGFα), transforming growth factor beta (TGFβ), basic fibroblastgrowth factor, vascular endothelial growth factor, epithelial growthfactor, nerve growth factor, platelet-derived growth factor, andvascular permeability factor. Vasodilators can include, for example,adenosine, adenosine derivatives, or combinations thereof,Anticoagulants include, but are not limited to, heparin, heparinderivatives, anti-factor Xa, anti-thrombin, aspirin, clopidgrel, orcombinations thereof.

In another aspect of the invention, methods are provided for promotingangiogenesis along or around a medical device by coating the device witha thyroid hormone, thyroid hormone analog, or polymeric form thereofaccording to the invention prior to inserting the device into a patient.The coating step can further include coating the device with one or morebiologically active substance, such as, but not limited to, a growthfactor, a vasodilator, an anti-coagulant, or combinations thereof.Examples of medical devices that can be coated with thyroid hormoneanalogs or polymeric forms according to the invention include stents,catheters, cannulas or electrodes.

In a further aspect, the invention provides methods for treating acondition amenable to treatment by inhibiting angiogenesis byadministering to a subject in need thereof an amount of ananti-angiogenesis agent effective for inhibiting angiogenesis.

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to, primary or metastatictumors, diabetic retinopathy, and related conditions. Examples of theanti-angiogenesis agents used for inhibiting angiogenesis are alsoprovided by the invention and include, but are not limited to,tetraiodothyroacetic acid (TETRAC), triiodothyroacetic acid (TRIAC),monoclonal antibody LM609, XT 199 or combinations thereof. Suchanti-angiogenesis agents can act at the cell surface to inhibit thepro-angiogenesis agents.

In one embodiment, the anti-angiogenesis agent is administered by aparenteral, oral, rectal, or topical mode, or combination thereof. Inanother embodiment, the anti-angiogenesis agent can be co-administeredwith one or more anti-angiogenesis therapies or chemotherapeutic agents.

In yet a further aspect, the invention provides compositions (i.e.,angiogenic agents) that include thyroid hormone, and analogs conjugatedto a polymer. The conjugation can be through a covalent or non-covalentbond, depending on the polymer. A covalent bond can occur through anester or anhydride linkage, for example. Examples of the thyroid hormoneanalogs are also provided by the instant invention and includelevothyroxine (T4), triiodothyronine (T3),3,5-dimethyl-4-(4′-hydroy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or 3,5-diiodothyropropionic acid (DITPA). In one embodiment, thepolymer can include, but is not limited to, polyvinyl alcohol, acrylicacid ethylene co-polymer, polylactic acid, or agarose.

In another aspect, the invention provides for pharmaceuticalformulations including the angiogenic agents according to the presentinvention in a pharmaceutically acceptable carrier. In one embodiment,the pharmaceutical formulations can also include one or morepharmaceutically acceptable excipients.

The pharmaceutical formulations according to the present invention canbe encapsulated or incorporated in a liposome, microparticle, orpolymer. The liposome or microparticle has a size of less than about 200nanometers. Any of the pharmaceutical formulations according to thepresent invention can be administered via parenteral, oral, rectal, ortopical means, or combinations thereof. In another embodiment, thepharmaceutical formulations can be co-administered to a subject in needthereof with one or more biologically active substances including, butnot limited to, growth factors, vasodilators, anti-coagulants, orcombinations thereof.

The details of one or more embodiments of the invention have been setforth in the accompanying description below. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural references unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All patents and publicationscited in this specification are incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effects of L-T4 and L-T3 on angiogenesis quantitated in thechick CAM assay. A, Control samples were exposed to PBS and additionalsamples to 1 nM T3 or 0.1 μmol/L T4 for 3 days. Both hormones causedincreased blood vessel branching in these representative images from 3experiments. B, Tabulation of mean±SEM of new branches formed fromexisting blood vessels during the experimental period drawn from 3experiments, each of which included 9 CAM assays. At the concentrationsshown, T3 and T4 caused similar effects (1.9-fold and 2.5-foldincreases, respectively, in branch formation). **P<0.001 by 1-way ANOVA,comparing hormone-treated with PBS-treated CAM samples.

FIG. 2. Tetrac inhibits stimulation of angiogenesis by T4 andagarose-linked T4 (T4-ag). A, A 2.5-fold increase in blood vessel branchformation is seen in a representative CAM preparation exposed to 0.1μmol/L T4 for 3 days. In 3 similar experiments, there was a 2.3-foldincrease. This effect of the hormone is inhibited by tetrac (0.1μmol/L), a T4 analogue shown previously to inhibit plasma membraneactions of T4.13 Tetrac alone does not stimulate angiogenesis (C). B,T4-ag (0.1 μmol/L) stimulates angiogenesis 2.3-fold (2.9-fold in 3experiments), an effect also blocked by tetrac. C, Summary of theresults of 3 experiments that examine the actions of tetrac, T4-ag, andT4 in the CAM assay. Data (means±SEM) were obtained from 10 images foreach experimental condition in each of 3 experiments. **P<0.001 byANOVA, comparing T4-treated and T4-agarose-treated samples withPBS-treated control samples.

FIG. 3. Comparison of the proangiogenic effects of FGF2 and T4. A,Tandem effects of T4 (0.05 μmol/L) and FGF2 (0.5 μg/mL) in submaximalconcentrations are additive in the CAM assay and equal the level ofangiogenesis seen with FGF2 (1 μg/mL in the absence of T4). B, Summaryof results from 3 experiments that examined actions of FGF2 and T4 inthe CAM assay (means±SEM) as in A. *P<0.05; **P<0.001, comparing resultsof treated samples with those of PBS-treated control samples in 3experiments.

FIG. 4. Effect of anti-FGF2 on angiogenesis caused by T4 or exogenousFGF2. A, FGF2 caused a 2-fold increase in angiogenesis in the CAM modelin 3 experiments, an effect inhibited by antibody (ab) to FGF2 (8 μg).T4 also stimulated angiogenesis 1.5-fold, and this effect was alsoblocked by FGF2 antibody, indicating that the action of thyroid hormonein the CAM model is mediated by an autocrine/paracrine effect of FGF2because T4 and T3 cause FGF2 release from cells in the CAM model (Table1). We have shown previously that a nonspecific IgG antibody has noeffect on angiogenesis in the CAM assay. B, Summary of results from 3CAM experiments that studied the action of FGF2-ab in the presence ofFGF2 or T4. *P<0.01; **P<0.001, indicating significant effects in 3experiments studying the effects of thyroid hormone and FGF2 onangiogenesis and loss of these effects in the presence of antibody toFGF2.

FIG. 5. Effect of PD 98059, a MAPK (ERK1/2) signal transduction cascadeinhibitor, on angiogenesis induced by T4, T3, and FGF2. A, Angiogenesisstimulated by T4 (0.1 μmol/L) and T3 (1 nmol/L) together is fullyinhibited by PD 98059 (3 μmol/L). B, Angiogenesis induced by FGF2 (1μg/mL) is also inhibited by PD 98059, indicating that the action of thegrowth factor is also dependent on activation of the ERK1/2 pathway. Inthe context of the experiments involving T4-agarose (T4-ag) and tetrac(FIG. 2) indicating that T4 initiates its proangiogenic effect at thecell membrane, results shown in A and B are consistent with 2 rolesplayed by MAPK in the proangiogenic action of thyroid hormone: ERK1/2transduces the early signal of the hormone that leads to FGF2elaboration and transduces the subsequent action of FGF2 onangiogenesis. C, Summary of results of 3 experiments, represented by Aand B, showing the effect of PD98059 on the actions of T4 and FGF2 inthe CAM model.

*P<0.01; **P<0.001, indicating results of ANOVA on data from 3experiments.

FIG. 6. T4 and FGF2 activate MAPK in ECV304 endothelial cells. Cellswere prepared in M199 medium with 0.25% hormone-depleted serum andtreated with T4 (0.1 μmol/L) for 15 minutes to 6 hours. Cells wereharvested and nuclear fractions prepared as described previously.Nucleoproteins, separated by gel electrophoresis, were immunoblottedwith antibody to phosphorylated MAPK (pERK1 and pERK2, 44 and 42 kDa,respectively), followed by a second antibody linked to aluminescence-detection system. A β-actin immunoblot of nuclear fractionsserves as a control for gel loading in each part of this figure. Eachimmunoblot is representative of 3 experiments. A, T4 causes increasedphosphorylation and nuclear translocation of ERK1/2 in ECV304 cells. Theeffect is maximal in 30 minutes, although the effect remains for ≧6hours. B, ECV304 cells were treated with the ERK1/2 activation inhibitorPD 98059 (PD; 30 μmol/L) or the PKC inhibitor CGP41251 (CGP; 100 nmol/L)for 30 minutes, after which 10⁻⁷M T4 was added for 15 minutes to cellsamples as shown. Nuclei were harvested, and this representativeexperiment shows increased phosphorylation (activation) of ERK1/2 by T4(lane 4), which is blocked by both inhibitors (lanes 5 and 6),suggesting that PKC activity is a requisite for MAPK activation by T4 inendothelial cells. C, ECV304 cells were treated with either T4 (10⁻⁷mol/L), FGF2 (10 ng/mL), or both agents for 15 minutes. The figure showspERK1/2 accumulation in nuclei with either hormone or growth factortreatment and enhanced nuclear pERK1/2 accumulation with both agentstogether.

FIG. 7. T4 increases accumulation of FGF2 cDNA in ECV304 endothelialcells. Cells were treated for 6 to 48 hours with T4 (10⁻⁷ mol/L) andFGF2 and GAPDH cDNAs isolated from each cell aliquot. The levels of FGF2cDNA, shown in the top blot, were corrected for variations in GAPDH cDNAcontent, shown in the bottom blot, and the corrected levels of FGF2 areillustrated below in the graph (mean±SE of mean; n=2 experiments). Therewas increased abundance of FGF2 transcript in RNA extracted from cellstreated with T4 at all time points. *P<0.05; **P<0.01, indicatingcomparison by ANOVA of values at each time point to control value.

FIG. 8. 7 Day Chick Embryo Tumor Growth Model. Illustration of the ChickChorioallantoic Membrane (CAM) model of tumor implant.

FIG. 9. T4 Stimulates 3D Wound Healing. Photographs of human dermalfibroblast cells exposed to T4 and control, according to the 3D WoundHealing Assay described herein.

FIG. 10. T4 Dose-Dependently Increases Wound Healing, Day 3. Asindicated by the graph, T4 increases wound healing (measured byoutmigrating cells) in a dose-dependent manner between concentrations of0.1 μM and 1.0 μM. This same increase is not seen in concentrations ofT4 between 1.0 μM and 3.0 μM.

FIG. 11. Effect of unlabeled T₄ and T₃ on ^(I-125)-T₄ binding topurified integrin. Unlabeled T₄ (10⁻⁴M to 10⁻¹¹M) or T₃ (10⁻⁴M to 10⁻⁸M)were added to purified αVβ3 integrin (2 μg/sample) and allowed toincubate for 30 min. at room temperature. Two microcuries of I-125labeled T₄ was added to each sample. The samples were incubated for 20min. at room temperature, mixed with loading dye, and run on a 5% Nativegel for 24 hrs. at 4° C. at 45 mÅ. Following electrophoresis, the gelswere wrapped in plastic wrap and exposed to film. ^(I-125)-T₄ binding topurified αVβ3 is unaffected by unlabeled T₄ in the range of 10⁻¹¹M to10⁻⁷M, but is competed out in a dose-dependent manner by unlabeled T₄ ata concentration of 10⁻⁶M. Hot T₄ binding to the integrin is almostcompletely displaced by 10⁻⁴M unlabeled T₄. T₃ is less effective atcompeting out T₄ binding to αVβ3, reducing the signal by 11%, 16%, and28% at 10⁻⁶M, 10⁻⁵M, and 10⁻⁴M T₃, respectively.

FIG. 12. Tetrac and an RGD containing peptide, but not an RGE containingpeptide compete out T₄ binding to purified αVβ3. A) Tetrac addition topurified αVβ3 reduces ^(I-125)-labeled T₄ binding to the integrin in adose dependent manner. 10⁻⁸M tetrac is ineffective at competing out hotT₄ binding to the integrin. The association of T₄ and αVβ3 was reducedby 38% in the presence of 10⁻⁷M tetrac and by 90% with 10⁻⁵M tetrac.Addition of an RGD peptide at 10⁻⁵M competes out T₄ binding to αVβ3.Application of 10⁻⁵M and 10⁻⁴M RGE peptide, as a control for the RGDpeptide, was unable to diminish hot T₄ binding to purified αVβ3. B)Graphical representation of the tetrac and RGD data from panel A. Datapoints are shown as the mean±S.D. for 3 independent experiments.

FIG. 13. Effects of the monoclonal antibody LM609 on T₄ binding to αVβ3.A) LM609 was added to αVβ3 at the indicated concentrations. One μg ofLM609 per sample reduces ^(I-125)-labeled T₄ binding to the integrin by52%. Maximal inhibition of T₄ binding to the integrin is reached whenconcentrations of LM609 are 2 μg per sample and is maintained withantibody concentrations as high as 8 μg. As a control for antibodyspecificity, 10 μg/sample Cox-2 mAB and 10 μg/sample mouse IgG wereadded to αVβ3 prior to incubation with T₄. B) Graphical representationof data from panel A. Data points are shown as the mean±S.D. for 3independent experiments.

FIG. 14. Effect of RGD, RGE, tetrac, and the mAB LM609 on T₄-inducedMAPK activation. A) CV-1 cells (50-70% confluency) were treated for 30min. with 10⁻⁷ M T₄ (10⁻⁷ M total concentration, 10⁻¹⁰ M freeconcentration. Selected samples were treated for 16 hrs with theindicated concentrations of either an RGD containing peptide, an RGEcontaining peptide, tetrac, or LM609 prior to the addition of T₄.Nuclear proteins ere separated by SDS-PAGE and immunoblotted withanti-phospho-MAPK (pERK1/2) antibody. Nuclear accumulation of pERK1/2 isdiminished in samples treated with 10⁻⁶M RGD peptide or higher, but notsignificantly altered in samples treated with 10⁻⁴ M RGE. pERK1/2accumulation is decreased 76% in CV1 cells treated with 10⁻⁶M tetrac,while 10⁻⁵M and higher concentrations of tetrac reduce nuclearaccumulation of pERK 1/2 to levels similar to the untreated controlsamples. The monoclonal antibody to αVβ3 LM609 decrease accumulation ofactivated MAPK in the nucleus when it is applied to CV1 cultures aconcentration of 1 μg/ml. B) Graphical representation of the data forRGD, RGE, and tetrac shown in panel A. Data points represent themean±S.D. for 3 separate experiments.

FIG. 15. Effects of siRNA to αV and β3 on T₄ induced MAPK activation.CV1 cells were transfected with siRNA (100 nM final concentration) toαV, β3, or αV and β3 together. Two days after transfection, the cellswere treated with 10⁻⁷M T₄. A) RT-PCR was performed from RNA isolatedfrom each transfection group to verify the specificity and functionalityof each siRNA. B) Nuclear proteins from each transfection were isolatedand subjected to SDS-PAGE.

FIG. 16. Inhibitory Effect of αVβ3 mAB (LM609) on T₄-stimulatedAngiogenesis in the CAM Model. A) Samples were exposed to PBS, T₄ (0.1μM), or T₄ plus 10 mg/ml LM609 for 3 days. Angiogenesis stimulated by T₄is substantially inhibited by the addition of the αVβ3 monoclonalantibody LM609. B) Tabulation of the mean±SEM of new branches formedfrom existing blood vessels during the experimental period. Data wasdrawn from 3 separate experiments, each containing 9 samples in eachtreatment group. C, D) Angiogenesis stimulated by T4 or FGF2 is alsoinhibited by the addition of the αVβ3 monoclonal antibody LM609 or XT199.

FIG. 17. Polymer Compositions of Thyroid Hormone Analogs—PolymerConjugation Through an Ester Linkage Using Polyvinyl Alcohol. In thispreparation commercially available polyvinyl alcohol (or relatedco-polymers) can be esterified by treatment with the acid chloride ofthyroid hormone analogs, namely the acid chloride form. Thehydrochloride salt is neutralized by the addition of triethylamine toafford triethylamine hydrochloride which can be washed away with waterupon precipitation of the thyroid hormone ester polymer form fordifferent analogs. The ester linkage to the polymer may undergohydrolysis in vivo to release the active pro-angiogenesis thyroidhormone analog.

FIG. 18. Polymer Compositions of Thyroid Hormone Analogs—PolymerConjugation Through an Anhydride Linkage Using Acrylic Acid EthyleneCo-polymer. This is similar to the previous polymer covalent conjugationhowever this time it is through an anhydride linkage that is derivedfrom reaction of an acrylic acid co-polymer. This anhydride linkage isalso susceptible to hydrolysis in vivo to release thyroid hormoneanalog. Neutralization of the hydrochloric acid is accomplished bytreatment with triethylamine and subsequent washing of the precipitatedpolyanhydride polymer with water removes the triethylamine hydrochloridebyproduct. This reaction will lead to the formation of Thyroid hormoneanalog acrylic acid co-polymer+triethylamine. Upon in vivo hydrolysis,the thyroid hormone analog will be released over time that can becontrolled plus acrylic acid ethylene Co-polymer.

FIG. 19. Polymer Compositions of Thyroid Hormone Analogs—Entrapment in aPolylactic Acid Polymer. Polylactic acid polyester polymers (PLA)undergo hydrolysis in vivo to the lactic acid monomer and this has beenexploited as a vehicle for drug delivery systems in humans. Unlike theprior two covalent methods where the thyroid hormone analog is linked bya chemical bond to the polymer, this would be a non-covalent method thatwould encapsulate the thyroid hormone analog into PLA polymer beads.This reaction will lead to the formation of Thyroid hormone analogcontaining PLA beads in water. Filter and washing will result in theformation of thyroid hormone analog containing PLA beads, which upon invivo hydrolysis will lead to the generation of controlled levels ofthyroid hormone plus lactic acid.

FIG. 20. Thyroid Hormone Analogs Capable of Conjugation with VariousPolymers. A-D show substitutions required to achieve various thyroidhormone analogs which can be conjugated to create polymeric forms ofthyroid hormone analogs of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described with references to the accompanying drawings, andas pointed out by the claims. For convenience, certain terms used in thespecification, examples and claims are collected here. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention pertains.

As used herein, the term “angiogenic agent” includes any compound orsubstance that promotes or encourages angiogenesis, whether alone or incombination with another substance. Examples include, but are notlimited to, T3, T4, T3 or T4-agarose, polymeric analogs of T3, T4,3,5-dimethyl-4-(4′-hydroy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or DITPA. In contrast, the terms “anti-angiogenesis agent” oranti-angiogenic agent” refer to any compound or substance that inhibitsor discourages angiogenesis, whether alone or in combination withanother substance. Examples include, but are not limited to, TETRAC,TRIAC, XT 199, and mAb LM609.

As used herein, the term “myocardial ischemia” is defined as aninsufficient blood supply to the heart muscle caused by a decreasedcapacity of the heart vessels. As used herein, the term “coronarydisease” is defined as diseases/disorders of cardiac function due to animbalance between myocardial function and the capacity of coronaryvessels to supply sufficient blood flow for normal function. Specificcoronary diseases/disorders associated with coronary disease which canbe treated with the compositions and methods described herein includemyocardial ischemia, angina pectoris, coronary aneurysm, coronarythrombosis, coronary vasospasm, coronary artery disease, coronary heartdisease, coronary occlusion and coronary stenosis.

As used herein the term “occlusive peripheral vascular disease” (alsoknown as peripheral arterial occlusive disorder) is a vasculardisorder-involving blockage in the carotid or femoral arteries,including the iliac artery. Blockage in the femoral arteries causes painand restricted movement. A specific disorder associated with occlusiveperipheral vascular disease is diabetic foot, which affects diabeticpatients, often resulting in amputation of the foot.

As used herein the terms “regeneration of blood vessels,”“angiogenesis,” “revascularization,” and “increased collateralcirculation” (or words to that effect) are considered as synonymous. Theterm “pharmaceutically acceptable” when referring to a natural orsynthetic substance means that the substance has an acceptable toxiceffect in view of its much greater beneficial effect, while the relatedthe term, “physiologically acceptable,” means the substance hasrelatively low toxicity. The term, “co-administered” means two or moredrugs are given to a patient at approximately the same time or in closesequence so that their effects run approximately concurrently orsubstantially overlap. This term includes sequential as well assimultaneous drug administration.

“Pharmaceutically acceptable salts” refers to pharmaceuticallyacceptable salts of thyroid hormone analogs, polymeric forms, andderivatives, which salts are derived from a variety of organic andinorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetra-alkyl ammonium, and the like; and when the molecule contains abasic functionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like can be used as the pharmaceutically acceptablesalt.

“Subject” includes living organisms such as humans, monkeys, cows,sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, culturedcells therefrom, and transgenic species thereof. In a preferredembodiment, the subject is a human. Administration of the compositionsof the present invention to a subject to be treated can be carried outusing known procedures, at dosages and for periods of time effective totreat the condition in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the age, sex, and weight of the subject,and the ability of the therapeutic compound to treat the foreign agentsin the subject. Dosage regimens can be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

“Administering” includes routes of administration which allow thecompositions of the invention to perform their intended function, e.g.,promoting angiogenesis. A variety of routes of administration arepossible including, but not necessarily limited to parenteral (e.g.,intravenous, intra-arterial, intramuscular, subcutaneous injection),oral (e.g., dietary), topical, nasal, rectal, or via slow releasingmicrocarriers depending on the disease or condition to be treated. Oral,parenteral and intravenous administration are preferred modes ofadministration. Formulation of the compound to be administered will varyaccording to the route of administration selected (e.g., solution,emulsion, gels, aerosols, capsule). An appropriate compositioncomprising the compound to be administered can be prepared in aphysiologically acceptable vehicle or carrier and optional adjuvants andpreservatives. For solutions or emulsions, suitable carriers include,for example, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media, sterile water, creams,ointments, lotions, oils, pastes and solid carriers. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers (See generally, Remington's PharmaceuticalScience, 16th Edition, Mack, Ed. (1980)).

“Effective amount” includes those amounts of pro-angiogenic oranti-angiogenic compounds which allow it to perform its intendedfunction, e.g., promoting or inhibiting angiogenesis inangiogenesis-related disorders as described herein. The effective amountwill depend upon a number of factors, including biological activity,age, body weight, sex, general health, severity of the condition to betreated, as well as appropriate pharmacokinetic properties. For example,dosages of the active substance may be from about 0.01 mg/kg/day toabout 500 mg/kg/day, advantageously from about 0.1 mg/kg/day to about100 mg/kg/day. A therapeutically effective amount of the activesubstance can be administered by an appropriate route in a single doseor multiple doses. Further, the dosages of the active substance can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation.

“Pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like which arecompatible with the activity of the compound and are physiologicallyacceptable to the subject. An example of a pharmaceutically acceptablecarrier is buffered normal saline (0.15M NaCl). The use of such mediaand agents for pharmaceutically active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the therapeutic compound, use thereof in the compositions suitablefor pharmaceutical administration is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

“Additional ingredients” include, but are not limited to, one or more ofthe following: excipients; surface active agents; dispersing agents;inert diluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed, e.g., in Remington's Pharmaceutical Sciences.

Compositions

Disclosed herein are angiogenic agents comprising thyroid hormones,analogs thereof, and polymer conjugations of the hormones and theiranalogs. The disclosed compositions can be used for promotingangiogenesis to treat disorders wherein angiogenesis is beneficial.Additionally, the inhibition of these thyroid hormones, analogs andpolymer conjugations can be used to inhibit angiogenesis to treatdisorders associated with such undesired angiogenesis. As used herein,the term “angiogenic agent” includes any compound or substance thatpromotes or encourages angiogenesis, whether alone or in combinationwith another substance. Examples include, but are not limited to, T3,T4, T3 or T4-agarose, polymeric analogs of T3, T4,3,5-dimethyl-4-(4′-hydroy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or DITPA.

Polymer conjugations are used to improve drug viability. While many oldand new therapeutics are well-tolerated, many compounds need advanceddrug discovery technologies to decrease toxicity, increase circulatorytime, or modify biodistribution. One strategy for improving drugviability is the utilization of water-soluble polymers. Variouswater-soluble polymers have been shown to modify biodistribution,improve the mode of cellular uptake, change the permeability throughphysiological barriers, and modify the rate of clearance through thebody. To achieve either a targeting or sustained-release effect,water-soluble polymers have been synthesized that contain drug moietiesas terminal groups, as part of the backbone, or as pendent groups on thepolymer chain.

Representative compositions of the present invention include thyroidhormone or analogs thereof conjugated to polymers. Conjugation withpolymers can be either through covalent or non-covalent linkages. Inpreferred embodiments, the polymer conjugation can occur through anester linkage or an anhydride linkage. An example of a polymerconjugation through an ester linkage using polyvinyl alcohol is shown inFIG. 17. In this preparation commercially available polyvinyl alcohol(or related co-polymers) can be esterified by treatment with the acidchloride of thyroid hormone analogs, including the acid chloride form.The hydrochloride salt is neutralized by the addition of triethylamineto afford triethylamine hydrochloride which can be washed away withwater upon precipitation of the thyroid hormone ester polymer form fordifferent analogs. The ester linkage to the polymer may undergohydrolysis in vivo to release the active pro-angiogenesis thyroidhormone analog.

An example of a polymer conjugation through an anhydride linkage usingacrylic acid ethylene co-polymer is shown in FIG. 18. This is similar tothe previous polymer covalent conjugation, however, this time it isthrough an anhydride linkage that is derived from reaction of an acrylicacid co-polymer. This anhydride linkage is also susceptible tohydrolysis in vivo to release thyroid hormone analog. Neutralization ofthe hydrochloric acid is accomplished by treatment with triethylamineand subsequent washing of the precipitated polyanhydride polymer withwater removes the triethylamine hydrochloride byproduct. This reactionwill lead to the formation of Thyroid hormone analog acrylic acidco-polymer+triethylamine. Upon in vivo hydrolysis, the thyroid hormoneanalog will be released over time that can be controlled plus acrylicacid ethylene Co-polymer.

Another representative polymer conjugation includes thyroid hormone orits analogs conjugated to polyethylene glycol (PEG). Attachment of PEGto various drugs, proteins and liposomes has been shown to improveresidence time and decrease toxicity. PEG can be coupled to activeagents through the hydroxyl groups at the ends of the chains and viaother chemical methods. Peg itself, however, is limited to two activeagents per molecule. In a different approach, copolymers of PEG andamino acids were explored as novel biomaterials which would retain thebiocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule and which could besynthetically designed to suit a variety of applications.

Another representative polymer conjugation includes thyroid hormone orits analogs in non-covalent conjugation with polymers. This is shown indetail in FIG. 19. A preferred non-covalent conjugation is entrapment ofthyroid hormone or analogs thereof in a polylactic acid polymer.Polylactic acid polyester polymers (PLA) undergo hydrolysis in vivo tothe lactic acid monomer and this has been exploited as a vehicle fordrug delivery systems in humans. Unlike the prior two covalent methodswhere the thyroid hormone analog is linked by a chemical bond to thepolymer, this would be a non-covalent method that would encapsulate thethyroid hormone analog into PLA polymer beads. This reaction will leadto the formation of Thyroid hormone analog containing PLA beads inwater. Filter and washing will result in the formation of thyroidhormone analog containing PLA beads, which upon in vivo hydrolysishydrolysis will lead to the generation of controlled levels of thyroidhormone plus lactic acid.

Furthermore, nanotechnology can be used for the creation of usefulmaterials and structures sized at the nanometer scale. The main drawbackwith biologically active substances is fragility. Nanoscale materialscan be combined with such biologically active substances to dramaticallyimprove the durability of the substance, create localized highconcentrations of the substance and reduce costs by minimizing losses.Therefore, additional polymeric conjugations include nano-particleformulations of thyroid hormones and analogs thereof. In such anembodiment, nano-polymers and nano-particles can be used as a marix forlocal delivery of thyrid hormone and its analogs. This will aid in timecontrolled delivery into the cellular and tissue target.

Compositions of the present invention include both thyroid hormone,analogs, and derivatives either alone or in covalent or non-covalentconjugation with polymers. Examples of representative analogs andderivatives are shown in FIG. 20, Tables A-D. Table A shows T2, T3, T4,and bromo-derivatives. Table B shows alanyl side chain modifications.Table C shows hydroxy groups, diphenyl ester linkages, andD-configurations. Table D shows tyrosine analogs.

The terms “anti-angiogenesis agent” or anti-angiogenic agent” refer toany compound or substance that inhibits or discourages angiogenesis,whether alone or in combination with another substance. Examplesinclude, but are not limited to, TETRAC, TRIAC, XT 199, and mAb LM609.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations inPromoting Angiogenesis

The pro-angiogenic effect of thyroid hormone analogs or polymeric formsdepends upon a non-genomic initiation, as tested by the susceptibilityof the hormonal effect to reduction by pharmacological inhibitors of theMAPK signal transduction pathway. Such results indicates that anotherconsequence of activation of MAPK by thyroid hormone is new blood vesselgrowth. The latter is initiated nongenomically, but of course, requiresa consequent complex gene transcription program. The ambientconcentrations of thyroid hormone are relatively stable. The CAM model,at the time we tested it, was thyroprival and thus may be regarded as asystem, which does not reproduce the intact organism.

The availability of a chick chorioallantoic membrane (CAM) assay forangiogenesis has provided a model in which to quantitate angiogenesisand to study possible mechanisms involved in the induction by thyroidhormone of new blood vessel growth. The present application discloses apro-angiogenic effect of T₄ that approximates that in the CAM model ofFGF2 and that can enhance the action of suboptimal doses of FGF2. It isfurther disclosed that the pro-angiogenic effect of the hormone isinitiated at the plasma membrane and is dependent upon activation by T₄of the MAPK signal transduction pathway. As provided above, methods fortreatment of occlusive peripheral vascular disease and coronarydiseases, in particular, the occlusion of coronary vessels, anddisorders associated with the occlusion of the peripheral vasculatureand/or coronary blood vessels are disclosed. Also disclosed arecompositions and methods for promoting angiogenesis and/or recruitingcollateral blood vessels in a patient in need thereof. The compositionsinclude an effective amount of Thyroid hormone analogs, polymeric forms,and derivatives. The methods involve the co-administration of aneffective amount of thyroid hormone analogs, polymeric forms, andderivatives in low, daily dosages for a week or more with other standardpro-angiogenesis growth factors, vasodilators, anticoagulants,thrombolytics or other vascular-related therapies.

The CAM assay has been used to validate angiogenic activity of a varietyof growth factors and compounds believed to promote angiogenesis. Forexample, T₄ in physiological concentrations was shown to bepro-angiogenic in this in vitro model and on a molar basis to have theactivity of FGF2. The presence of PTU did not reduce the effect of T₄,indicating that de-iodination of T₄ to generate T₃ was not aprerequisite in this model. A summary of the pro-angiogenesis effects ofvarious thyroid hormone analogs is listed in Table 1.

TABLE 1 Pro-angiogenesis Effects of Various Thyroid Hormone Analogs inthe CAM Model TREATMENT ANGIOGENESIS INDEX PBS (Control)  89.4 ± 9.3DITPA (0.01 uM)  133.0 ± 11.6 DITPA (0.1 uM)  167.3 ± 12.7 DITPA (0.2mM) 117.9 ± 5.6 GC-1 (0.01 uM)  169.6 ± 11.6 GC-1 (0.1 uM) 152.7 ± 9.0T4 agarose (0.1 uM) 195.5 ± 8.5 T4 (0.1 uM) 143.8 ± 7.9 FGF2 (1 ug) 155± 9 n = 8 per group

The appearance of new blood vessel growth in this model requires severaldays, indicating that the effect of thyroid hormone was wholly dependentupon the interaction of the nuclear receptor for thyroid hormone (TR)with the hormone. Actions of iodothyronines that require intranuclearcomplexing of TR with its natural ligand, T₃, are by definition,genomic, and culminate in gene expression. On the other hand, thepreferential response of this model system to T₄—rather than T₃, thenatural ligand of TR—raised the possibility that angiogenesis might beinitiated nongenomically at the plasma membrane by T₄ and culminate ineffects that require gene transcription. Non-genomic actions of T₄ havebeen widely described, are usually initiated at the plasma membrane andmay be mediated by signal transduction pathways. They do not requireintranuclear ligand of iodothyronine and TR, but may interface with ormodulate gene transcription. Non-genomic actions of steroids have alsobeen well described and are known to interface with genomic actions ofsteroids or of other compounds. Experiments carried out with T₄ andtetrac or with agarose-T₄ indicated that the pro-angiogenic effect of T₄indeed very likely was initiated at the plasma membrane. Tetrac blocksmembrane-initiated effects of T₄, but does not, itself, activate signaltransduction. Thus, it is a probe for non-genomic actions of thyroidhormone. Agarose-T₄ is thought not to gain entry to the cell interiorand has been used to examine models for possible cell surface-initiatedactions of the hormone.

In part, this invention provides compositions and methods for promotingangiogenesis in a subject in need thereof. Conditions amenable totreatment by promoting angiogenesis include, for example, occlusiveperipheral vascular disease and coronary diseases, in particular, theocclusion of coronary vessels, and disorders associated with theocclusion of the peripheral vasculature and/or coronary blood vessels,erectile dysfunction, stroke, and wounds. Also disclosed arecompositions and methods for promoting angiogenesis and/or recruitingcollateral blood vessels in a patient in need thereof. The compositionsinclude an effective amount of polymeric forms of thyroid hormoneanalogs and derivatives and an effective amount of an adenosine and/ornitric oxide donor. The compositions can be in the form of a sterile,injectable, pharmaceutical formulation that includes an angiogenicallyeffective amount of thyroid hormone-like substance and adenosinederivatives in a physiologically and pharmaceutically acceptablecarrier, optionally with one or more excipients.

Myocardial Infarction

A major reason for heart failure following acute myocardial infarctionis an inadequate response of new blood vessel formation, i.e.,angiogenesis. Thyroid hormone and its analogs are beneficial in heartfailure and stimulate coronary angiogenesis. The methods of theinvention include, in part, delivering a single treatment of a thyroidhormone analog at the time of infarction either by direct injection intothe myocardium, or by simulation of coronary injection by intermittentaortic ligation to produce transient isovolumic contractions to achieveangiogenesis and/or ventricular remodeling.

Accordingly, in one aspect the invention features methods for treatingocclusive vascular disease, coronary disease, myocardial infarction,ischemia, stroke, and/or peripheral artery vascular disorders bypromoting angiogenesis by administering to a subject in need thereof anamount of a polymeric form of thyroid hormone, or an analog thereateffective for promoting angiogenesis.

Examples of polymeric forms of thyroid hormone analogs are also providedherein and can include triiodothyronine (T3), levothyroxine (T4),(GC-1), or 3,5-diiodothyropropionic acid (DITPA) conjugated to polyvinylalcohol, acrylic acid ethylene co-polymer, polylactic acid, or agarose.

The methods also involve the co-administration of an effective amount ofthyroid hormone-like substance and an effective amount of an adenosineand/or NO donor in low, daily dosages for a week or more. One or bothcomponents can be delivered locally via catheter. Thyroid hormoneanalogs, and derivatives in vivo can be delivered to capillary bedssurrounding ischemic tissue by incorporation of the compounds in anappropriately sized liposome or microparticle. Thyroid hormone analogs,polymeric forms and derivatives can be targeted to ischemic tissue bycovalent linkage with a suitable antibody.

The method may be used as a treatment to restore cardiac function aftera myocardial infarction. The method may also be used to improve bloodflow in patients with coronary artery disease suffering from myocardialischemia or inadequate blood flow to areas other than the heartincluding, for example, occlusive peripheral vascular disease (alsoknown as peripheral arterial occlusive disease), or erectiledysfunction.

Wound Healing

Wound angiogenesis is an important part of the proliferative phase ofhealing. Healing of any skin wound other than the most superficialcannot occur without angiogenesis. Not only does any damaged vasculatureneed to be repaired, but the increased local cell activity necessary forhealing requires an increased supply of nutrients from the bloodstream.Moreover, the endothelial cells which form the lining of the bloodvessels are important in themselves as organizers and regulators ofhealing.

Thus, angiogenesis provides a new microcirculation to support thehealing wound. The new blood vessels become clinically visible withinthe wound space by four days after injury. Vascular endothelial cells,fibroblasts, and smooth muscle cells all proliferate in coordination tosupport wound granulation. Simultaneously, re-epithelialization occursto reestablish the epithelial cover. Epithelial cells from the woundmargin or from deep hair follicles migrate across the wound andestablish themselves over the granulation tissue and provisional matrix.Growth factors such as keratinocyte growth factor (KGF) mediate thisprocess. Several models (sliding versus rolling cells) ofepithelialization exist.

As thyroid hormones regulate metabolic rate, when the metabolism slowsdown due to hypothyroidism, wound healing also slows down. The role oftopically applied thyroid hormone analogs or polymeric forms in woundhealing therefore represents a novel strategy to accelerate woundhealing in diabetics and in non-diabetics with impaired wound healingabilities. Topical administration can be in the form of attachment to aband-aid. Additionally, nano-polymers and nano-particles can be used asa marix for local delivery of thyrid hormone and its analogs. This willaid in time controlled delivery into the cellular and tissue target.

Accordingly, another embodiment of the invention features methods fortreating wounds by promoting angiogenesis by administering to a subjectin need thereof an amount of a polymeric form of thyroid hormone, or ananalog thereof, effective for promoting angiogenesis. For details, seeExample 9.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations inInhibiting Angiogenesis

The invention also provides, in another part, compositions and methodsfor inhibiting angiogenesis in a subject in need thereof. Conditionsamenable to treatment by inhibiting angiogenesis include, for example,primary or metastatic tumors and diabetic retinopathy. The compositionscan include an effective amount of TETRAC, TRIAC or mAb LM609. Thecompositions can be in the form of a sterile, injectable, pharmaceuticalformulation that includes an anti-angiogenically effective amount of ananti-angiogenic substance in a physiologically and pharmaceuticallyacceptable carrier, optionally with one or more excipients.

In a further aspect, the invention provides methods for treating acondition amenable to treatment by inhibiting angiogenesis byadministering to a subject in need thereof an amount of ananti-angiogenesis agent effective for inhibiting angiogenesis.

Examples of the anti-angiogenesis agents used for inhibitingangiogenesis are also provided by the invention and include, but are notlimited to, tetraiodothyroacetic acid (TETRAC), triiodothyroacetic acid(TRIAC), monoclonal antibody LM609, or combinations thereof. Suchanti-angiogenesis agents can act at the cell surface to inhibit thepro-angiogenesis agents.

Cancer-Related New Blood Vessel Growth

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to, primary or metastatictumors. In such a method, compounds which inhibit the thyroidhormone-induced angiogenic effect are used to inhibit angiogenesis.Details of such a method is illustrated in Example 12.

Diabetic Retinopathy

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to diabetic retinopathy, andrelated conditions. In such a method, compounds which inhibit thethyroid hormone-induced angiogenic effect are used to inhibitangiogenesis. Details of such a method is illustrated in Examples 8A andB.

It is known that proliferative retinopathy induced by hypoxia (ratherthan diabetes) depends upon alphaV (αV) integrin expression (E Chavakiset al., Diabetologia 45:262-267, 2002). It is proposed herein thatthyroid hormone action on a specific integrin alphaVbeta-3 (αVβ3) ispermissive in the development of diabetic retinopathy. Integrin αVβ3 isidentified herein as the cell surface receptor for thyroid hormone.Thyroid hormone, its analogs, and polymer conjugations, act via thisreceptor to induce angiogenesis.

Methods of Treatment

Thyroid hormone analogs, polymeric forms, and derivatives can be used ina method for promoting angiogenesis in a patient in need thereof. Themethod involves the co-administration of an effective amount of thyroidhormone analogs, polymeric forms, and derivatives in low, daily dosagesfor a week or more. The method may be used as a treatment to restorecardiac function after a myocardial infarction. The method may also beused to improve blood flow in patients with coronary artery diseasesuffering from myocardial ischemia or inadequate blood flow to areasother than the heart, for example, peripheral vascular disease, forexample, peripheral arterial occlusive disease, where decreased bloodflow is a problem.

The compounds can be administered via any medically acceptable meanswhich is suitable for the compound to be administered, including oral,rectal, topical or parenteral (including subcutaneous, intramuscular andintravenous) administration. For example, adenosine has a very shorthalf-life. For this reason, it is preferably administered intravenously.However, adenosine A.sub.2 agonists have been developed which have muchlonger half-lives, and which can be administered through other means.Thyroid hormone analogs, polymeric forms, and derivatives can beadministered, for example, intravenously, oral, topical, intranasaladministration.

In some embodiments, the thyroid hormone analogs, polymeric forms, andderivatives are administered via different means.

The amounts of the thyroid hormone, its analogs, polymeric forms, andderivatives required to be effective in stimulating angiogenesis will,of course, vary with the individual being treated and is ultimately atthe discretion of the physician. The factors to be considered includethe condition of the patient being treated, the efficacy of theparticular adenosine A.sub.2 receptor agonist being used, the nature ofthe formulation, and the patient's body weight. Occlusion-treatingdosages of thyroid hormone analogs or its polymeric forms, andderivatives are any dosages that provide the desired effect.

Formulations

The compounds described above are preferably administered in aformulation including thyroid hormone analogs or its polymeric forms,and derivatives together with an acceptable carrier for the mode ofadministration. Any formulation or drug delivery system containing theactive ingredients, which is suitable for the intended use, as aregenerally known to those of skill in the art, can be used. Suitablepharmaceutically acceptable carriers for oral, rectal, topical orparenteral (including subcutaneous, intraperitoneal, intramuscular andintravenous) administration are known to those of skill in the art. Thecarrier must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Formulations suitable for parenteral administration conveniently includesterileaqueous preparation of the active compound, which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound of formula (I), which upon dilution withan appropriate solvent give a solution suitable for parentaladministration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contentsof which are hereby incorporated by reference.

In one embodiment, the thyroid hormone analogs or its polymeric forms,and adenosine derivatives can be formulated into a liposome ormicroparticle, which is suitably sized to lodge in capillary bedsfollowing intravenous administration. When the liposome or microparticleis lodged in the capillary beds surrounding ischemic tissue, the agentscan be administered locally to the site at which they can be mosteffective. Suitable liposomes for targeting ischemic tissue aregenerally less than about 200 nanometers and are also typicallyunilamellar vesicles, as disclosed, for example, in U.S. Pat. No.5,593,688 to Baldeschweiler, entitled “Liposomal targeting of ischemictissue,” the contents of which are hereby incorporated by reference.

Preferred microparticles are those prepared from biodegradable polymers,such as polyglycolide, polylactide and copolymers thereof. Those ofskill in the art can readily determine an appropriate carrier systemdepending on various factors, including the desired rate of drug releaseand the desired dosage.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered to the inside of a tissue lumen and the active compoundsreleased over time as the polymer degrades. If desirable, the polymerichydrogels can have microparticles or liposomes which include the activecompound dispersed therein, providing another mechanism for thecontrolled release of the active compounds.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

The formulations can optionally include additional components, such asvarious biologically active substances such as growth factors (includingTGF-.beta., basic fibroblast growth factor (FGF2), epithelial growthfactor (EGF), transforming growth factors .alpha. and .beta. (TGF alpha.and beta.), nerve growth factor (NGF), platelet-derived growth factor(PDGF), and vascular endothelial growth factor/vascular permeabilityfactor (VEGF/VPF)), antiviral, antibacterial, anti-inflammatory,immuno-suppressant, analgesic, vascularizing agent, and cell adhesionmolecule.

In addition to the aforementioned ingredients, the formulations mayfurther include one or more optional accessory ingredient(s) utilized inthe art of pharmaceutical formulations, e.g., diluents, buffers,flavoring agents, binders, surface active agents, thickeners,lubricants, suspending agents, preservatives (including antioxidants)and the like.

Materials & Methods

Reagents:

All reagents were chemical grade and purchased from Sigma Chemical Co.(St. Louis, Mo.) or through VWR Scientific (Bridgeport, N.J.). Cortisoneacetate, bovine serum albumin (BSA) and gelatin solution (2% type B frombovine skin) were purchased from Sigma Chemical Co. Fertilized chickeneggs were purchased from Charles River Laboratories, SPAFAS AvianProducts & Services (North Franklin, Conn.). T4,3,5,3′-triiodo-L-thyronine (T3), tetraiodothyroacetic acid (tetrac),T4-agarose, and 6-N-propyl-2-thiouracil (PTU) were obtained from Sigma;PD 98059 from Calbiochem; and CGP41251 was a gift from Novartis Pharma(Basel, Switzerland). Polyclonal anti-FGF2 and monoclonal anti-β-actinwere obtained from Santa Cruz Biotechnology and human recombinant FGF2from Invitrogen. Polyclonal antibody to phosphorylated ERK1/2 was fromNew England Biolabs and goat anti-rabbit IgG from DAKO.

Chorioallantoic membrane (CAM) Model of Angiogenesis: In vivoNeovascularization was examined by methods described previously. 9-12Ten-day-old chick embryos were purchased from SPAFAS (Preston, Conn.)and incubated at 37° C. with 55% relative humidity. A hypodermic needlewas used to make a small hole in the shell concealing the air sac, and asecond hole was made on the broad side of the egg, directly over anavascular portion of the embryonic membrane that was identified bycandling. A false air sac was created beneath the second hole by theapplication of negative pressure at the first hole, causing the CAM toseparate from the shell. A window approximately 1.0 cm 2 was cut in theshell over the dropped CAM with a small-crafts grinding wheel (Dremel,division of Emerson Electric Co.), allowing direct access to theunderlying CAM. FGF2 (1 μg/mL) was used as a standard proangiogenicagent to induce new blood vessel branches on the CAM of 10-day-oldembryos. Sterile disks of No. 1 filter paper (Whatman International)were pretreated with 3 mg/mL cortisone acetate and 1 mmol/L PTU and airdried under sterile conditions. Thyroid hormone, hormone analogues, FGF2or control solvents, and inhibitors were then applied to the disks andthe disks allowed to dry. The disks were then suspended in PBS andplaced on growing CAMs. Filters treated with T4 or FGF2 were placed onthe first day of the 3-day incubation, with antibody to FGF2 added 30minutes later to selected samples as indicated. At 24 hours, the MAPKcascade inhibitor PD 98059 was also added to CAMs topically by means ofthe filter disks.

Microscopic Analysis of CAM Sections:

After incubation at 37° C. with 55% relative humidity for 3 days, theCAM tissue directly beneath each filter disk was resected from controland treated CAM samples. Tissues were washed 3× with PBS, placed in35-mm Petri dishes (Nalge Nunc), and examined under an SV6stereomicroscope (Zeiss) at ×50 magnification. Digital images of CAMsections exposed to filters were collected using a 3-charge-coupleddevice color video camera system (Toshiba) and analyzed with Image-Prosoftware (Media Cybernetics). The number of vessel branch pointscontained in a circular region equal to the area of each filter diskwere counted. One image was counted in each CAM preparation, andfindings from 8 to 10 CAM preparations were analyzed for each treatmentcondition (thyroid hormone or analogues, FGF2, FGF2 antibody, PD 98059).In addition, each experiment was performed 3 times. The resultingangiogenesis index is the mean±SEM of new branch points in each set ofsamples.

FGF2 Assays:

ECV304 endothelial cells were cultured in M199 medium supplemented with10% fetal bovine serum. ECV304 cells (10⁶ cells) were plated on 0.2%gel-coated 24-well plates in complete medium overnight, and the cellswere then washed with serum-free medium and treated with T4 or T3 asindicated. After 72 hours, the supernatants were harvested and assaysfor FGF performed without dilution using a commercial ELISA system (R&DSystems).

MAPK Activation:

ECV304 endothelial cells were cultured in M199 medium with 0.25%hormone-depleted serum 13 for 2 days. Cells were then treated with T4(10⁻⁷ mol/L) for 15 minutes to 6 hours. In additional experiments, cellswere treated with T4 or FGF2 or with T4 in the presence of PD 98059 orCGP41251. Nuclear fractions were pre-pared from all samples by ourmethod reported previously, the proteins separated by polyacrylamide gelelectrophoresis, and transferred to membranes for immunoblotting withantibody to phosphorylated ERK 1/2. The appearance of nuclearphosphorylated ERK1/2 signifies activation of these MAPK isoforms by T4.

Reverse Transcription-Polymerase Chain Reaction:

Confluent ECV304 cells in 10-cm plates were treated with T4 (10⁻⁷ mol/L)for 6 to 48 hours and total RNA extracted using guanidiniumisothiocyanate (Biotecx Laboratories). RNA (1 μg) was subjected toreverse transcription-polymerase chain reaction (RT-PCR) using theAccess RT-PCR system (Promega). Total RNA was reverse transcribed intocDNA at 48° C. for 45 minutes, then denatured at 94° C. for 2 minutes.Second-strand synthesis and PCR amplification were performed for 40cycles with denaturation at 94° C. for 30 s, annealing at 60° C. for 60s, and extension at 68° C. for 120 s, with final ex-tension for 7minutes at 68° C. after completion of all cycles. PCR primers for FGF2were as follows: FGF2 sense strand 5′-TGGTATGTGGCACTGAAACG-3′ (SEQ IDNO:1), antisense strand 5′CTCAATGACCTGGCGAAGAC-3′ (SEQ ID NO:2); thelength of the PCR product was 734 bp. Primers for GAPDH included thesense strand 5′-AAGGTCATCCCTGAGCTGAACG-3′ (SEQ ID NO:3), and antisensestrand 5′-GGGTGTCGCTGTTGAAGTCAGA-3′ (SEQ ID NO:4); the length of the PCRproduct was 218 bp. The products of RT-PCR were separated byelectrophoresis on 1.5% agarose gels and visualized with ethidiumbromide. The target bands of the gel were quantified using LabImagesoftware (Kapelan), and the value for [FGF2/GAPDH]×10 calculated foreach time point.

Statistical Analysis:

Statistical analysis was performed by 1-way ANOVA comparing experimentalwith control samples.

In Vivo Angiogenesis in Matrigel FGF₂ or Cancer Cell Lines Implant inMice: In Vivo Murine Angiogenesis Model:

The murine matrigel model will be conducted according to previouslydescribed methods (Grant et al., 1991; Okada et al., 1995) and asimplemented in our laboratory (Powel et al., 2000). Briefly, growthfactor free matrigel (Becton Dickinson, Bedford Mass.) will be thawedovernight at 4° C. and placed on ice. Aliquots of matrigel will beplaced into cold polypropylene tubes and FGF2, thyroid hormone analogsor cancer cells (1×10⁶ cells) will be added to the matrigel. Matrigelwith Saline, FGF2, thyroid hormone analogs or cancer cells will besubcutaneously injected into the ventral midline of the mice. At day 14,the mice will be sacrificed and the solidified gels will be resected andanalyzed for presence of new vessels. Compounds A-D will be injectedsubcutaneously at different doses. Control and experimental gel implantswill be placed in a micro centrifuge tube containing 0.5 ml of celllysis solution (Sigma, St. Louis, Mo.) and crushed with a pestle.Subsequently, the tubes will be allowed to incubate overnight at 4° C.and centrifuged at 1,500×g for 15 minutes on the following day. A 200 μlaliquot of cell lysate will be added to 1.3 ml of Drabkin's reagentsolution (Sigma, St. Louis, Mo.) for each sample. The solution will beanalyzed on a spectrophotometer at a 540 nm. The absorption of light isproportional to the amount of hemoglobin contained in the sample.

Tumor Growth and Metastasis—Chick Chorioallantoic Membrane (CAM) Modelof Tumor Implant:

The protocol is as previously described (Kim et al., 2001). Briefly,1×10⁷ tumor cells will be placed on the surface of each CAM (7 day oldembryo) and incubated for one week. The resulting tumors will be excisedand cut into 50 mg fragments. These fragments will be placed onadditional 10 CAMs per group and treated topically the following daywith 25 μl of compounds (A-D) dissolved in PBS. Seven days later, tumorswill then be excised from the egg and tumor weights will be determinedfor each CAM. FIG. 8 is a diagrammatic sketch showing the steps involvedin the in vivo tumor growth model in the CAM.

The effects of TETRAC, TRIAC, and thyroid hormone antagonists on tumorgrowth rate, tumor angiogenesis, and tumor metastasis of cancer celllines can be determined.

Tumor Growth and Metastasis—Tumor Xenograft Model in Mice:

The model is as described in our publications by Kerr et al., 2000; VanWaes et al., 2000; Ali et al., 2001; and Ali et al., 2001, each of whichis incorporated herein by reference in its entirety). The anti-cancerefficacy for TETRAC, TRIAC, and other thyroid hormone antagonists atdifferent doses and against different tumor types can be determined andcompared.

Tumor Growth and Metastasis—Experimental Model of Metastasis:

The model is as described in our recent publications (Mousa, 2002;Amirkhosravi et al., 2003a and 2003b, each of which is incorporated byreference herein in its entirety). Briefly, B16 murine malignantmelanoma cells (ATCC, Rockville, Md.) and other cancer lines will becultured in RPMI 1640 (Invitrogen, Carlsbad, Calif.), supplemented with10% fetal bovine serum, penicillin and streptomycin (Sigma, St. Louis,Mo.). Cells will be cultured to 70% confluency and harvested withtrypsin-EDTA (Sigma) and washed twice with phosphate buffered saline(PBS). Cells will be re-suspended in PBS at a concentration of either2.0×10⁵ cells/ml for experimental metastasis. Animals: C57/BL6 mice(Harlan, Indianapolis, Ind.) weighing 18-21 grams will be used for thisstudy. All procedures are in accordance with IACUC and institutionalguidelines. The anti-cancer efficacy for TETRAC, TRIAC, and otherthyroid hormone antagonists at different doses and against differenttumor types can be determined and compared.

Effect of Thyroid Hormone Analogues on Angiogenesis.

T4 induced significant increase in angiogenesis index (fold increaseabove basal) in the CAM model. T3 at 0.001-1.0 μM or T4 at 0.1-1.0 μMachieved maximal effect in producing 2-2.5 fold increase in angiogenesisindex as compared to 2-3 fold increase in angiogenesis index by 1 μg ofFGF2 (Table 1 and FIGS. 1 a and 1 b). The effect of T4 in promotingangiogenesis (2-2.5 fold increase in angiogenesis index) was achieved inthe presence or absence of PTU, which inhibit T4 to T3 conversion. T3itself at 91-100 nM)-induced potent pro-angiogenic effect in the CAMmodel. T4 agarose produced similar pro-angiogenesis effect to thatachieved by T4. The pro-angiogenic effect of either T4 or T4-agarose was100% blocked by TETRAC or TRIAC.

Enhancement of Pro-Angiogenic Activity of FGF2 by Sub-MaximalConcentrations of T₄.

The combination of T4 and FGF2 at sub-maximal concentrations resulted inan additive increase in the angiogenesis index up to the same level likethe maximal pro-angiogenesis effect of either FGF2 or T4 (FIG. 2).

Effects of MAPK Cascade Inhibitors on the Pro-Angiogenic Actions of T₄and FGf2 n the CAM Model.

The pro-angiogenesis effect of either T4 or FGF2 was totally blocked byPD 98059 at 0.8-8 μg (FIG. 3).

Effects of Specific Integrin αVβ3 Antagonists on the Pro-AngiogenicActions of T₄ and FGf2 n the CAM Model.

The pro-angiogenesis effect of either T4 or FGF2 was totally blocked bythe specific monoclonal antibody LM609 at 10 μg (FIGS. 4 a and 4 b).

The CAM assay has been used to validate angiogenic activity of a varietyof growth factors and other promoters or inhibitors of angiogenesis(2-9). In the present studies, T₄ in physiological concentrations wasshown to be pro-angiogenic, with comparable activity to that of FGF2.The presence of PTU did not reduce the effect of T₄, indicating thatde-iodination of T₄ to generate T₃ was not a prerequisite in this model.Because the appearance of new blood vessel growth in this model requiresseveral days, we assumed that the effect of thyroid hormone was totallydependent upon the interaction of the nuclear receptor for thyroidhormone (TR). Actions of iodothyronines that require intranuclearcomplexing of TR with its natural ligand, T₃, are by definition,genomic, and culminate in gene expression. On the other hand, thepreferential response of this model system to T₄—rather than T₃, thenatural ligand of TR raised the possibility that angiogenesis might beinitiated non-gnomically at the plasma membrane by T₄ and culminate ineffects that require gene transcription. Non-genomic actions of T₄ havebeen widely described, are usually initiated at the plasma membrane andmay be mediated by signal transduction pathways. They do not requireintranuclear ligand binding of iodothyronine and TR, but may interfacewith or modulate gene transcription. Non-genomic actions of steroidshave also been well-described and are known to interface with genomicactions of steroids or of other compounds. Experiments carried out withT₄ and tetrac or with agarose-T₄ indicated that the pro-angiogeniceffect of T₄ indeed very likely was initiated at the plasma membrane. Wehave shown elsewhere that tetrac blocks membrane-initiated effects ofT₄, but does not, itself, activate signal transduction. Thus, it is aprobe for non-genomic actions of thyroid hormone. Agarose-T₄ is thoughtnot to gain entry to the cell interior and has been used by us andothers to examine models for possible cell surface-initiated actions ofthe hormone.

These results suggest that another consequence of activation of MAPK bythyroid hormone is new blood vessel growth. The latter is initiatednongenomically, but of course requires a consequent complex genetranscription program.

The ambient concentrations of thyroid hormone are relatively stable. TheCAM model, at the time we tested it, was thyroprival and thus may beregarded as a system, which does not reproduce the intact organism. Wepropose that circulating levels of T₄ serve, with a variety of otherregulators, to modulate the sensitivity of vessels to endogenousangiogenic factors, such as VEGF and FGF2.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLES Example 1

Effect of Thyroid Hormone on Angiogenesis: As seen in FIG. 1A andsummarized in FIG. 1B, both L-T4 and L-T3 enhanced angiogenesis in theCAM assay. T4, at a physiologic total concentration in the medium of 0.1μmol/L, increased blood vessel branch formation by 2.5-fold (P<0.001).T3 (1 nmol/L) also stimulated angiogenesis 2-fold. The possibility thatT4 was only effective because of conversion of T4 to T3 by cellular5′-monodeiodinase was ruled out by the finding that the deiodinaseinhibitor PTU had no inhibitory effect on angiogenesis produced by T4.PTU was applied to all filter disks used in the CAM model. Thus, T4 andT3 promote new blood vessel branch formation in a CAM model that hasbeen standardized previously for the assay of growth factors.

Example 2

Effects of T4-Agarose and Tetrac: We have shown previously thatT4-agarose stimulates cellular signal transduction pathways initiated atthe plasma membrane in the same manner as T4 and that the actions of T4and T4-agarose are blocked by a deaminated iodothyronine analogue,tetrac, which is known to inhibit binding of T4 to plasma membranes. Inthe CAM model, the addition of tetrac (0.1 μmol/L) inhibited the actionof T4 (FIG. 2A), but tetrac alone had no effect on angiogenesis (FIG.2C). The action of T4-agarose, added at a hormone concentration of 0.1μmol/L, was comparable to that of T4 in the CAM model (FIG. 2B), and theeffect of T4-agarose was also inhibited by the action of tetrac (FIG.2B; summarized in 2C).

Example 3

Enhancement of Proangiogenic Activity of FGF2 by a SubmaximalConcentration of T4: Angiogenesis is a complex process that usuallyrequires the participation of polypeptide growth factors. The CAM assayrequires at least 48 hours for vessel growth to be manifest; thus, theapparent plasma membrane effects of thyroid hormone in this model arelikely to result in a complex transcriptional response to the hormone.Therefore, we determined whether FGF2 was involved in the hormoneresponse and whether the hormone might potentiate the effect ofsubphysiologic levels of this growth factor. T4 (0.05 μmol/L) and FGF2(0.5 μg/mL) individually stimulated angiogenesis to a modest degree(FIG. 3). The angiogenic effect of this submaximal concentration of FGF2was enhanced by a subphysiologic concentration of T4 to the level causedby 1.0 μg FGF2 alone. Thus, the effects of submaximal hormone and growthfactor concentrations appear to be additive. To define more preciselythe role of FGF2 in thyroid hormone stimulation of angiogenesis, apolyclonal antibody to FGF2 was added to the filters treated with eitherFGF2 or T4, and angiogenesis was measured after 72 hours. FIG. 4demonstrates that the FGF2 antibody inhibited angiogenesis stimulatedeither by FGF2 or by T4 in the absence of exogenous FGF2, suggestingthat the T4 effect in the CAM assay was mediated by increased FGF2expression. Control IgG antibody has no stimulatory or inhibitory effectin the CAM assay.

Example 4

Stimulation of FGF2 Release From Endothelial Cells by Thyroid Hormone:Levels of FGF2 were measured in the media of ECV304 endothelial cellstreated with either T4 (0.1 μmol/L) or T3 (0.01 μmol/L) for 3 days. Asseen in the Table 2, T3 stimulated FGF2 concentration in the medium3.6-fold, whereas T4 caused a 1.4-fold increase. This finding indicatesthat thyroid hormone may enhance the angiogenic effect of FGF2, at leastin part, by increasing the concentration of growth factor available toendothelial cells.

TABLE 2 Effect of T4 and T3 on Release of FGF2 From ECV304 EndothelialCells Cell Treatment FGF2 (pg/mL/10⁶ cells) Control 27.7 ± 3.1 T3 (0.01μmol/L)  98.8 ± 0.5* T3 + PD 98059 (2 μmol/L) 28.4 ± 3.2 T3 + PD 98059(20 μmol/L) 21.7 ± 3.5 T4 (0.1 μmol/L)  39.2 ± 2.8† T4 + PD 98059 (2μmol/L) 26.5 ± 4.5 T4 + PD 98059 (20 μmol/L) 23.2 ± 4.8 *P < 0.001,comparing T3-treated samples with control samples by ANOVA; †P < 0.05,comparing T4-treated samples with control samples by ANOVA.

Example 5

Role of the ERK1/2 Signal Transduction Pathway in Stimulation ofAngiogenesis by Thyroid Hormone and FGF2: A pathway by which T4 exerts anongenomic effect on cells is the MAPK signal transduction cascade,specifically that of ERK1/2 activation. We know that T4 enhances ERK1/2activation by epidermal growth factor. The role of the MAPK pathway instimulation by thyroid hormone of FGF2 expression was examined by theuse of PD 98059 (2 to 20 μmol/L), an inhibitor of ERK1/2 activation bythe tyrosine-threonine kinases MAPK kinase-1 (MEK1) and MEK2. The datain the Table demonstrate that PD 98059 effectively blocked the increasein FGF2 release from ECV304 endothelial cells treated with either T4 orT3. Parallel studies of ERK1/2 inhibition were performed in CAM assays,and representative results are shown in FIG. 5. A combination of T3 andT4, each in physiologic concentrations, caused a 2.4-fold increase inblood vessel branching, an effect that was completely blocked by 3μmol/L PD 98059 (FIG. 5A). FGF2 stimulation of branch formation(2.2-fold) was also effectively blocked by this inhibitor of ERK1/2activation (FIG. 5B). Thus, the proangiogenic effect of thyroid hormonebegins at the plasma membrane and involves activation of the ERK1/2pathway to promote FGF2 release from endothelial cells. ERK1/2activation is again required to transduce the FGF2 signal and cause newblood vessel formation.

Example 6

Action of Thyroid Hormone and FGF2 on MAPK Activation Stimulation ofphosphorylation and nuclear translocation of ERK1/2 MAPKs was studied inECV304 cells treated with T4 (10⁻⁷ mol/L) for 15 minutes to 6 hours. Theappearance of phosphorylated ERK1/2 in cell nuclei occurred within 15minutes of T4 treatment, reached a maximal level at 30 minutes, and wasstill apparent at 6 hours (FIG. 6A). This effect of the hormone wasinhibited by PD 98059 (FIG. 6B), a result to be expected because thiscompound blocks the phosphorylation of ERK1/2 by MAPK kinase. Thetraditional protein kinase C (PKC)-α, PKC-β, and PKC-γ inhibitorCGP41251 also blocked the effect of the hormone on MAPK activation inthese cells, as we have seen with T4 in other cell lines. Thyroidhormone enhances the action of several cytokines and growth factors,such as interferon-γ13 and epidermal growth factor. In ECV304 cells, T4enhanced the MAPK activation caused by FGF2 in a 15-minute co incubation(FIG. 6C). Applying observations made in ECV304 cells to the CAM model,we propose that the complex mechanism by which the hormone inducesangiogenesis includes endothelial cell release of FGF2 and enhancementof the autocrine effect of released FGF2 on angiogenesis.

Example 7

RT-PCR in ECV304 Cells Treated With Thyroid Hormone: The final questionaddressed in studies of the mechanism of the proangiogenic action of T4was whether the hormone may induce FGF2 gene expression. Endothelialcells were treated with T4 (10⁻⁷ mol/L) for 6 to 48 hours, andRT-PCR-based estimates of FGF2 and GAPDH RNA (inferred from cDNAmeasurements; FIG. 7) were performed. Increase in abundance of FGF2cDNA, corrected for GAPDH content, was apparent by 6 hours of hormonetreatment and was further enhanced by 48 hours.

Example 8A

Retinal Neovascularization model in mice (diabetic and non-diabetic): Toassess the pharmacologic activity of a test article on retinalneovascularization, Infant mice are exposed to a high oxygen environmentfor 7 days and allowed to recover, thereby stimulating the formation ofnew vessels on the retina. Test articles are evaluated to determine ifretinal neovascularization is suppressed. The retinas are examined withhematoxylin-eosin staining and with at least one stain, whichdemonstrates neovascularization (usually a Selectin stain). Other stains(such as PCNA, PAS, GFAP, markers of angiogenesis, etc.) can be used. Asummary of the model is below:

Animal Model

-   -   Infant mice (P7) and their dams are placed in a hyper-oxygenated        environment (70-80%) for 7 days.    -   On P12, the mice are removed from the oxygenated environment and        placed into a normal environment    -   Mice are allowed to recover for 5-7 days.    -   Mice are then sacrificed and the eyes collected.    -   Eyes are either frozen or fixed as appropriate    -   The eyes are stained with appropriate histochemical stains    -   The eyes are stained with appropriate immunohistochemical stains    -   Blood, serum, or other tissues can be collected    -   Eyes, with special reference to microvascular alterations, are        examined for any and all findings. Neovascular growth will be        semi quantitatively scored. Image analysis is also available.

Example 8B Thyroid Hormone and Diabetic Retinopathy

A protocol disclosed in J de la Cruz et al., J Pharmacol Exp Ther280:454-459, 1997, is used for the administration of Tetrac to rats thathave streptozotocin (STZ)-induced experimental diabetes and diabeticretinopathy. The endpoint is the inhibition by Tetrac of the appearanceof proliferative retinopathy (angiogenesis).

Example 9

In vitro human epithelial and fibroblast wound healing: The in vitro2-dimensional wound healing method is as described in Mohamed S,Nadijcka D, Hanson, V. Wound healing properties of cimetidine in vitro.Drug Intell Clin Pharm 20: 973-975; 1986, incorporated herein byreference in its entirety. Additionally, a 3-dimensional wound healingmethod already established in our Laboratory will be utilized in thisstudy (see below). Data show potent stimulation of wound healing bythyroid hormone.

In Vitro 3D Wound Healing Assay of Human Dermal Fibroblast Cells:

Step 1: Prepare Contracted Collagen Gels:

-   -   1) Coat 24-well plate with 350 ul 2% BSA at RT for 2 hr,    -   2) 80% confluent NHDF (normal human dermal fibroblast cells,        Passage 5-9) are trypsinized and neutralized with growth medium,        centrifuge and wash once with PBS    -   3) Prepare collagen-cell mixture, mix gently and always on ice:

Stock solution Final Concentration 5 × DMEC 1 × DMEM 3 mg/ml vitrogen 2mg/ml ddH2O optimal NHDF 2 × 10~5 cells/ml FBS 1%

-   -   4) Aspire 2% BSA from 24 well plate, add collagen-cell mixture        350 ul/well, and incubate the plate in 37° C. CO2 incubator.    -   5) After 1 hr, add DMEM+5% FBS medium 0.5 ml/well, use a 10 ul        tip Detach the collagen gel from the edge of each well, then        incubate for 2 days. The fibroblast cells will contract the        collagen gel        Step 2: Prepare 3D Fibrin Wound Clot and Embed Wounded Collagen        Culture    -   1) Prepare fibrinogen solution (1 mg/ml) with or without testing        regents. 350 ul fibrinogen solution for each well in eppendorf        tube.

Stock solution Final Concentration 5 × DMEC 1 × DMEM Fibrinogen 1 mg/mlddH2O optimal testing regents optimal concentration FBS 1% or 5%

-   -   2) Cut each contracted collagen gel from middle with scissors.        Wash the gel with PBS and transfer the gel to the center of each        well of 24 well plate    -   3) Add 1.5 ul of human thrombin (0.25 U/ul) to each tube, mix        well and then add the solution around the collagen gel, the        solution will polymerize in 10 mins.

After 20 mins, add DMEM+1% (or 5%) FBS with or without testing agent,450 ul/well and incubate the plate in 37° C. CO2 incubator for up to 5days. Take pictures on each day.

In Vivo Wound Healing in Diabetic Rats:

Using an acute incision wound model in diabetic rats, the effects ofthyroid hormone analogs and its conjugated forms are tested. The rate ofwound closure, breaking strength analyses and histology are performedperiodically on days 3-21.

Example 10

Rodent Model of Myocardial Infarction: The coronary artery ligationmodel of myocardial infarction is used to investigate cardiac functionin rats. The rat is initially anesthetized with xylazine and ketamine,and after appropriate anesthesia is obtained, the trachea is intubatedand positive pressure ventilation is initiated. The animal is placedsupine with its extremities loosely taped and a median sternotomy isperformed. The heart is gently exteriorized and a 6-O suture is firmlytied around the left anterior descending coronary artery. The heart israpidly replaced in the chest and the thoracotomy incision is closedwith a 3-O purse string suture followed by skin closure with interruptedsutures or surgical clips. Animals are placed on a temperature regulatedheating pad and closely observed during recovery. Supplemental oxygenand cardiopulmonary resuscitation are administered if necessary. Afterrecovery, the rat is returned to the animal care facility. Such coronaryartery ligation in the rat produces large anterior wall myocardialinfarctions. The 48 hr. mortality for this procedure can be as high as50%, and there is variability in the size of the infarct produced bythis procedure. Based on these considerations, and prior experience, toobtain 16-20 rats with large infarcts so that the two models of thyroidhormone delivery discussed below can be compared, approximately 400 ratsare required.

These experiments are designed to show that systemic administration ofthyroid hormone either before or after coronary artery ligation leads tobeneficial effects in intact animals, including the extent ofhemodynamic abnormalities assessed by echocardiography and hemodynamicmeasurements, and reduction of infarct size. Outcome measurements areproposed at three weeks post-infarction. Although some rats may have noinfarction, or only a small infarction is produced, these rats can beidentified by normal echocardiograms and normal hemodynamics (LVend-diastolic pressure<8 mm Hg).

Thyroid Hormone Delivery

There are two delivery approaches. In the first, thyroid hormone isdirectly injected into the peri-infarct myocardium. As the demarcationbetween normal and ischemic myocardium is easily identified during theacute open chest occlusion, this approach provides sufficient deliveryof hormone to detect angiogenic effects.

Although the first model is useful in patients undergoing coronaryartery bypass surgery, and constitutes proof of principle that one localinjection induces angiogenesis, a broader approach using a second modelcan also be used. In the second model, a catheter retrograde is placedinto the left ventricle via a carotid artery in the anesthetized ratprior to inducing myocardial infarction. Alternatively, a direct needlepuncture of the aorta, just above the aortic valve, is performed. Theintracoronary injection of the thyroid hormone is then simulated byabruptly occluding the aorta above the origin of the coronary vesselsfor several seconds, thereby producing isovolumic contractions. Thyroidhormone is then injected into the left ventricle or aorta immediatelyafter aortic constriction. The resulting isovolumic contractions propelblood down the coronary vessels perfusing the entire myocardium withthyroid hormone. This procedure can be done as many times as necessaryto achieve effectiveness. The number of injections depends on the dosesused and the formation of new blood vessels.

Echocardiography:

A method for obtaining 2-D and M-mode echocardiograms in unanesthetizedrats has been developed. Left ventricular dimensions, function, wallthickness and wall motion can be reproducibly and reliably measured. Themeasurement are carried out in a blinded fashion to eliminate bias withrespect to thyroid hormone administration.

Hemodynamics:

Hemodynamic measurements are used to determine the degree of leftventricular impairment. Rats are anesthetized with isoflurane. Throughan incision along the right anterior neck, the right carotid artery andthe right jugular vein are isolated and cannulated with a pressuretransducing catheter (Millar, SPR-612, 1.2 Fr). The followingmeasurements are then made: heart rate, systolic and diastolic BP, meanarterial pressure, left ventricular systolic and end-diastolic pressure,and + and −dP/dt. Of particular utility are measurements of leftventricular end-diastolic pressure, progressive elevation of whichcorrelates with the degree of myocardial damage.

Infarct Size:

Rats are sacrificed for measurement of infarct size using TTCmethodology.

Morphometry

Microvessel density [microvessels/mm²] will be measured in the infarctarea, peri-infarct area, and in the spared myocardium opposing theinfarction, usually the posterior wall. From each rat, 7-10 microscopichigh power fields [×400] with transversely sectioned myocytes will bedigitally recorded using Image Analysis software. Microvessels will becounted by a blinded investigator. The microcirculation will be definedas vessels beyond third order arterioles with a diameter of 150micrometers or less, supplying tissue between arterioles and venules. Tocorrect for differences in left ventricular hypertrophy, microvesseldensity will be divided by LV weight corrected for body weight.Myocardium from sham operated rats will serves as controls.

Example 11

Effects of the αvβ3 antagonists on the pro-angiogenesis effect of T4 orFGF2: The αvβ3 inhibitor LM609 totally inhibited both FGF2 or T4-inducedpro-angiogenic effects in the CAM model at 10 micrograms (FIG. 16).

Example 12 Inhibition of Cancer-Related New Blood Vessel Growth

A protocol disclosed in J. Bennett, Proc Natl Acad Sci USA 99:2211-2215,2002, is used for the administration of tetraiodothyroacetic (Tetrac) toSCID mice that have received implants of human breast cancer cells(MCF-7). Tetrac is provided in drinking water to raise the circulatinglevel of the hormone analog in the mouse model to 10-6 M. The endpointis the inhibitory action of tetrac on angiogenesis about the implantedtumors.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method for inhibiting angiogenesis in apatient, said method comprising administering to a subject in needthereof an effective amount of tetraiodothyroacetic acid (TETRAC)conjugated via a covalent bond to a polymer, wherein said polymer ispolyglycolide, polylactic acid, or co-polymers thereof, wherein saidpolymer is formulated into a nanoparticle, wherein the nanoparticle isless than 200 nanometers, and wherein the tetraiodothyroacetic acid(TETRAC) acts at the cell membrane level to inhibit pro-angiogenesisagents.
 2. The method of claim 1, wherein the mode of administration ofthe anti-angiogenesis agent is parenteral, oral, rectal, topical, orcombinations thereof.
 3. The method of claim 1, wherein theanti-angiogenesis agent is co-administered with one or more otheranti-angiogenesis therapies or chemotherapeutic agents.